Melanoma metastasis is driven by extensive intratumoral heterogeneity and phenotypic plasticity, yet how clonal identity relates to transcriptional programs during metastasis remains unclear. Here, we applied MeRLin, a single-cell lineage tracing platform, to dissect the clonal and transcriptional heterogeneity of metastatic melanoma in a patient-derived spontaneous metastasis model. Clonal analyses revealed hierarchical structures during tumor progression, with a subset of lineages from primary tumors consistently enriched across metastatic sites, supporting a model of polyclonal seeding followed by selective expansion of pre-existing highly metastatic subpopulations. Single-cell transcriptomic profiling identified two major metastatic subpopulations of distinct transcriptional programs, characterized by neural crest stem cell-like and lipid metabolism signatures. Both programs were enriched for invasion-associated genes and maintained across organs through distinct regulatory networks. Spatial mapping by barcode RNA-FISH linked these transcriptional states to their tissue context and showed that OLFML3 expression partially co-localized with a dominant subpopulation at the tumor-liver interface, marking the invasive fronts of metastatic growth. Together, these findings establish a framework in which clonal identity, transcriptional state, and spatial organization jointly shape metastatic melanoma progression.

BACKGROUND: Most advanced melanomas initially respond to targeted therapy but eventually relapse. Increasing evidence suggests that drug-tolerant persister cells can adopt a reversible drug-refractory state and represent a key driver of therapeutic resistance. METHODS: We developed MeRLin, a lineage tracing platform that integrates cellular barcoding, single-cell transcriptomic profiling, RNA fluorescence in situ hybridization, and computational analyses to track clonal and transcriptional dynamics in a patient-derived melanoma model during prolonged targeted therapy. Longitudinal analyses enabled the characterization of clonal fates, transcriptional states, and spatial organization of persister populations. RESULTS: Clonal dynamics showed that persister subpopulations initially responded to therapy, persisted through minimal residual disease, and expanded during tumor recurrence. Four persister-associated transcriptional states characterized by stress-like, lipid metabolism, PI3K signaling, and extracellular matrix remodeling programs were associated with persister populations arising from minor pre-treatment subpopulations under sustained drug pressure. Spatial transcriptomic analyses revealed structured spatial organization of these programs and suggested coordinated autocrine and paracrine interactions among persister states. Targeted barcode RNA fluorescence in situ hybridization enabled spatial mapping of clonal identity and gene expression, revealing in situ co-localization of a dominant resistant clone marked by SLC2A1 expression. CONCLUSIONS: Together, MeRLin provides a robust framework for dissecting cancer heterogeneity and characterizing persister subpopulations. Our findings demonstrate that melanoma recurrence is associated with diverse, spatially organized persister states linked to adaptive transcriptional programs.

Although BRAF/MEK inhibitor (BRAFi/MEKi) therapy initially shows high efficacy in patients with BRAF V600 E/K cutaneous melanoma, resistance develops in over 75% of cases. We tested robustness of the umbrella trial strategy in this population by analyzing relationships between genomic status of a gene and associated downstream consequences at the protein level. The results revealed weak relationships among mutations, copy-number amplification, and protein expression and activation. An in vivo compound repurposing screen using 11 clinically relevant agents from an NCI-portfolio with pan-RTK, non-RTK, and/or PI3K-mTOR specificity identified dasatinib as most capable of restoring sensitivity to BRAFi/MEKi in patient-derived xenograft (PDX) models originating from tumors that had progressed on BRAFi ± MEKi. High baseline expression of BRAFi/MEKi resistance-associated proteins (e.g., AXL, YAP, HSP70, and phospho-AKT) was predictive of the response to BRAFi/MEKi + dasatinib combination therapy. These findings suggest that adding dasatinib may help overcome resistance and restore anti-tumor activity in patients with BRAFi/MEKi-refractory cutaneous melanoma.

Supplemental Figure S1: LAI remodels the lipidome of melanoma cells. Supplemental Figure S2: LAI induces UGCG, and UGCG inhibition augments DC661 cytotoxicity, and this effect is not seen by blocking cholesterol synthesis and key earlier steps of autophagy. Supplemental Figure S3: LAI increases the formation of GMM in cancer cells. Supplemental Figure S4: UGCG inhibition synergistically augments LAI induced cytotoxicity and abrogates GMM formation without elevating ceramide levels. Supplemental Figure S5: LAI induces UGCG-associated GMM formation in PDX cells, a combination of DC661 and eliglustat impairs tumor growth in a therapy-resistant PDX tumor model.

Supplemental Table S1: Patient Characteristics by UGCG expression defined Risk Groups in the Learning Dataset; Supplemental Table S2: Patient Characteristics by UGCG expression defined Risk Groups in the Validation Dataset (Cirenajwis et al., 2015)

Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in >60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression serve as a mechanism of intrinsic early/adaptive CDK4i/6i resistance. ALM cells that have acquired CDK4i/6i resistance following chronic treatment exposure also exhibit hyperactivation of the MAPK pathway. MEK and/or ERK inhibition increases CDK4i/6i efficacy against therapy naïve and CDK4i/6i-resistant AM cells in xenograft and patient-derived xenograft (PDX) models and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach for patients with metastatic ALM to improve outcomes.

Preclinical activity of MFF targeting

Resistance to combination BRAF/MEK inhibitor (BRAFi/MEKi) therapy arises in nearly every patient with BRAF melanoma, despite promising initial responses. Achieving cures in this expanding BRAFi/MEKi-resistant cohort represents one of the greatest challenges to the field; few experience additional durable benefit from immunotherapy and no alternative therapies exist. To better personalize therapy in cancer patients to address therapy relapse, umbrella trials have been initiated whereby genomic sequencing of a panel of potentially actionable targets guide therapy selection for patients; however, the superior efficacy of such approaches remains to be seen. We here test the robustness of the umbrella trial rationale by analyzing relationships between genomic status of a gene and the downstream consequences at the protein level of related pathway, which find poor relationships between mutations, copy number amplification, and protein level. To profile candidate therapeutic strategies that may offer clinical benefit in the context of acquired BRAFi/MEKi resistance, we established a repository of patient-derived xenograft models from heavily pretreated patients with resistance to BRAFi/MEKi and/or immunotherapy (R-PDX). With these RPDXs, we executed in vivo compound repurposing screens using 11 FDA-approved agents from an NCIportfolio with pan-RTK, non-RTK and/or PI3K-mTOR specificity. We identify dasatinib as capable of restoring BRAFi/MEKi antitumor efficacy in ~70% of R-PDX tested. A systems-biology analysis indicates elevated baseline protein expression of canonical drivers of therapy resistance (e.g., AXL, YAP, HSP70, phosphoAKT) as predictive of MAPKi/dasatinib sensitivity. We therefore propose that dasatinib-based MAPKi therapy may restore antitumor efficacy in patients that have relapsed to standard-of-care therapy by broadly targeting proteins critical in melanoma therapy escape. Further, we submit that this experimental PDX paradigm could potentially improve preclinical evaluation of therapeutic modalities and augment our ability to identify biomarker-defined patient subsets that may respond to a given clinical trial. INTRODUCTION: There has been immense progress in the available treatment for patients with metastatic melanoma, with 14 therapies FDA-approved since 2011. Vertical targeting of the mitogen-activated protein kinase (MAPK) pathway with the use of dual BRAF/MEK inhibitor (BRAFi/MEKi) therapy for patients with BRAF mutant melanoma, as well as immune checkpoint inhibitor-based strategies available for all genotypes of melanoma have each improved the overall survival of patients with advanced disease. However, long-term responses beyond 5 years are only seen in 25-35% of patients, due to the persistence of therapy-resistant subpopulations of melanoma cells that employ diverse mechanisms of escape to drive intrinsic and acquired resistance, including cell stateand signaling pathway-plasticity. In the way of cell state plasticity that enable therapy escape, melanoma cells can dedifferentiate to occupy a variety of alternative identities including a neural crest stem cell-like identity (1), an invasive identity, and a stress cell identity (2). Regarding signaling pathway plasticity, a growing body of evidence identifies hyperactivation of compensatory signaling through the PI3K/AKT/mTORC1 (3), SRC (4), and STAT3 (5) pathways via upregulation of receptor tyrosine kinases (RTKs) including insulin growth factor receptor 1 (IGFR-1) (6), AXL (7), PDGFRβ (8), EGFR (9), c-MET (10), and HER3 (ERBB3) (11). However, efforts to target these diverse resistance mechanisms have not resulted in a novel approved-therapy regimen to address patients who have already relapsed to standard-of-care (SOC) therapies. Due to the immense tumoral heterogeneity across melanoma patients and the need for more personalized therapy strategies, “master” protocols have been created to provide medications better tailored to the unique genetic makeup of a given patient’s tumor (12). The national cancer institute (NCI) is currently testing a master protocol in the NCI molecular analysis for therapy choice (MATCH) trial, whereby cancer patients are provided therapy regimens based on the genetic changes found in their tumors (13, 14). Although highly innovative and geared to better address tumor heterogeneity, it remains to be seen how effective this strategy will be. Trials such as the NCI-MATCH are built upon a set of scientific assumptions including a) a correlation between an activating mutation in a pathway node and elevated activation of that respective pathway (e.g., PI3K mutations correlate with activation of PI3K/AKT/mTOR at the protein level), b) copy number increases of a gene correlate with the protein expression of that gene (e.g., tumors with EGFR amplification would exhibit increased EGFR protein expression), and c) tumors that harbor a given mutation are more sensitive to inhibitors that target the related pathway (e.g., tumors that harbor AKT mutations should be more sensitive to an mTOR inhibitor). There are genomic alterations that predict sensitivity to certain compounds, including BRAF mutations for V600E/K selective BRAF inhibitors (e.g., vemurafenib) in melanoma patients and EGFR mutations for EGFR inhibitors (e.g., gefitinib) in patients with advanced lung adenocarcinoma, however how robust the relationship between genomic status and antitumor efficacy of a cognate inhibitor is remains to be fully understood. Here, we aim to develop a therapy regimen for melanoma patients that have already relapsed to SOC therapies and for which no effective therapy regimens remain. Using a genomically, transcriptionally, and proteomically characterized set of patient-derived xenograft (PDX) models of metastatic melanoma, the relationship between mutation, copy number status, and total-/phospho-protein expression was analyzed, revealing a poor correlation. These analyses were expanded to cutaneous melanoma patient samples in the TCGA where reverse-phase protein array, copy number variation, and mutational information is available, which also corroborated the poor relationship between genomic and protein status. To develop an effective salvage therapy strategy, we developed a PDX therapy repurposing screen to investigate the potential for a triple combination including BRAFi/MEKi and a third inhibitor against the PI3K/AKT/mTOR pathway or a panRTK inhibitors to increase the overall survival relative to BRAFi/MEKi treatment alone. Relationships between gene and protein status with therapy efficacy reveal benefit in assessing totaland phospho-protein expression when determining tailored therapy strategies for patients. Of the 11 triple combinations screened, the most effective consisted of BRAFi/MEKi plus dasatinib. Sensitivity analysis and scRNAseq suggest the robust efficacy of the dasatinib combination in 14 models of BRAFi/MEKi-resistant PDX models relies on the broad target profile of dasatinib capable of suppressing diverse melanoma subpopulations critical in minimal residual disease and therapy escape. RESULTS: BRAFi/MEKi resistant tumors display elevated RTK/MAPK/PI3K/mTOR activity The majority of patients with advanced BRAF mutant melanoma will progress while on BRAFi/MEKi therapy. Further, four out of five of these patients show no long-term benefit from immunotherapy (i.e., antiPD-1, anti-CTLA-4) due to diverse mechanisms that confer cross-resistance across different therapeutic modalities (15). Therefore, there is an unmet need for durable secondand/or third-line (salvage) therapy strategies for patients that have already relapsed on standard-of-care (SOC) strategies. Although the list of reported resistance mechanisms to BRAFi/MEKi continues to grow (16-23), they predominately center around reactivation of the MAPK pathway and the hyperactivation of parallel survival signaling through the PI3K/AKT/mTOR pathway via elevated receptor tyrosine kinase (RTKs) and non-receptor kinase activity. In agreement, reverse-phase protein array (RPPA) analyses of 94 treatment-naïve, 22 BRAFi-resistant, and 16 BRAFi/MEKi-resistant patient-derived xenograft (PDX) models reveal a heterogeneous enrichment in various total and phospho-proteins involved in RTK, PI3K/AKT/mTOR, and MAPK signaling (Figure 1A). IGFBP2, phospho-HER3 (Y1289), phospho-cMET (Y1235), phospho-MEK (S217/S221), and phosphoERK1/2 (T202/Y204) levels were significantly elevated amongst BRAFiand BRAFi/MEKi-resistant PDX models relative to therapy naïve models. AXL, a dogmatic marker of a therapy resistance melanoma cell state was not significantly increased in BRAFiand BRAFi/MEKi-resistant models. However, reduced MITF expression was observed, suggesting melanoma models with acquired resistance occupy a de-differentiated cell state, as been previously suggested. Of note, multiple nodes of the PI3K/AKT/mTOR pathway (e.g., phospho-AKT T308/S473, phospho-mTOR S2448, phospho-4E-BP1 T37/T46/S65, phospho-S6 S235/S236/S240/S244) were not significantly different between therapy resistant PDX models and therapynaïve models (Figure 1A). To begin our efforts to develop an effective salvage strategy for patients that have relapsed SOC therapy, we designed a custom drug screen using 11 Food and Drug Administration (FDA)-approved compounds from a National Cancer Institute (NCI) drug repository that possess pan-RTK, non-RTK and/or PI3K-mTOR specificity (Figure 1B, 1C, Table 1). Each of the compounds used in this study have been previously tested in advanced melanoma patients, however not in combination with BRAFi/MEKi and not in the context of patients who have previously relapsed to BRAFi or BRAFi/MEKi in a stage II or greater trial. With the goal to identify a three-drug cocktail consisting of BRAFi/MEKi in combination with a third compound that possesses robust secondand/or third-line efficacy, we initially selected a discovery set of 5 R-PDX models derived from patients that have previous

MFF1 synthetic peptides used in this study

Peptidyl mimicry of MFF-VDAC1 complex

Anti-glioma activity of MFF peptidomimetic

Resistance to combination BRAF/MEK inhibitor (BRAFi/MEKi) therapy arises in nearly every patient with BRAF V600E/K melanoma, despite promising initial responses. Achieving cures in this expanding BRAFi/MEKi-resistant cohort represents one of the greatest challenges to the field; few experience additional durable benefit from immunotherapy and no alternative therapies exist. To better personalize therapy in cancer patients to address therapy relapse, umbrella trials have been initiated whereby genomic sequencing of a panel of potentially actionable targets guide therapy selection for patients; however, the superior efficacy of such approaches remains to be seen. We here test the robustness of the umbrella trial rationale by analyzing relationships between genomic status of a gene and the downstream consequences at the protein level of related pathway, which find poor relationships between mutations, copy number amplification, and protein level. To profile candidate therapeutic strategies that may offer clinical benefit in the context of acquired BRAFi/MEKi resistance, we established a repository of p atient- d erived x enograft models from heavily pretreated patients with resistance to BRAFi/MEKi and/or immunotherapy (R-PDX). With these R-PDXs, we executed in vivo compound repurposing screens using 11 FDA-approved agents from an NCI-portfolio with pan-RTK, non-RTK and/or PI3K-mTOR specificity. We identify dasatinib as capable of restoring BRAFi/MEKi antitumor efficacy in ∼70% of R-PDX tested. A systems-biology analysis indicates elevated baseline protein expression of canonical drivers of therapy resistance (e.g., AXL, YAP, HSP70, phospho-AKT) as predictive of MAPKi/dasatinib sensitivity. We therefore propose that dasatinib-based MAPKi therapy may restore antitumor efficacy in patients that have relapsed to standard-of-care therapy by broadly targeting proteins critical in melanoma therapy escape. Further, we submit that this experimental PDX paradigm could potentially improve preclinical evaluation of therapeutic modalities and augment our ability to identify biomarker-defined patient subsets that may respond to a given clinical trial. SINGLE SENTENCE SUMMARY Broad target inhibition effective as a salvage strategy in BRAF/MEK inhibitor-acquired resistance PDX

Requirements of MFF regulation of tumor cell death

Mutant peptides derived from MFF1 peptide #8 used in this study

Morphology of MFF targeting in tumor cells

Clinico-pathological characteristics of non-small cell lung cancer (NSCLC) patient series used in this study (n=72)

MFF-VDAC1 complex in cancer

AbstractLysosomal autophagy inhibition (LAI) with hydroxychloroquine or DC661 can enhance cancer therapy, but tumor regrowth is common. To elucidate LAI resistance, proteomics and immunoblotting demonstrated that LAI induced lipid metabolism enzymes in multiple cancer cell lines. Lipidomics showed that LAI increased cholesterol, sphingolipids, and glycosphingolipids. These changes were associated with striking levels of GM1+ membrane microdomains (GMM) in plasma membranes and lysosomes. Inhibition of cholesterol/sphingolipid metabolism proteins enhanced LAI cytotoxicity. Targeting UDP-glucose ceramide glucosyltransferase (UGCG) synergistically augmented LAI cytotoxicity. Although UGCG inhibition decreased LAI-induced GMM and augmented cell death, UGCG overexpression led to LAI resistance. Melanoma patients with high UGCG expression had significantly shorter disease-specific survival. The FDA-approved UGCG inhibitor eliglustat combined with LAI significantly inhibited tumor growth and improved survival in syngeneic tumors and a therapy-resistant patient-derived xenograft. These findings nominate UGCG as a new cancer target, and clinical trials testing UGCG inhibition in combination with LAI are warranted.Significance:We discovered UGCG-dependent lipid remodeling drives resistance to LAI. Targeting UGCG with a drug approved for a lysosomal storage disorder enhanced LAI antitumor activity without toxicity. LAI and UGCG inhibition could be tested clinically in multiple cancers.This article is highlighted in the In This Issue feature, p. 247

Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells(1-7). Molecular differences in rare individual cells in the initial population enable certain cells to become resistant to therapy(7-9); however, comparatively little is known about the variability in the resistance outcomes. Here we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing, to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically and functionally distinct resistant types. These resistant types are largely predetermined by molecular differences between cells before drug addition and not by extrinsic factors. Changes in the dose and type of drug can switch the resistant type of an initial cell, resulting in the generation and elimination of certain resistant types. Samples from patients show evidence for the existence of these resistant types in a clinical context. We observed diversity in resistant types across several single-cell-derived cancer cell lines and cell types treated with a variety of drugs. The diversity of resistant types as a result of the variability in intrinsic cell states may be a generic feature of responses to external cues.